Assays have been developed that employ fluorescent labels to facilitate detection of the analyte. Fluorescence is generally the result of a three-stage process. In the first stage, energy is supplied by an external source, such as an incandescent lamp or a laser, and absorbed by the fluorescent compound, creating an excited electronic singlet state. In the second stage, the excited state exists for a finite time during which the fluorescent compound undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. During this time, the energy of the excited state is partially dissipated, yielding a relaxed state from which fluorescence emission originates. The third stage is the fluorescence emission stage wherein energy is emitted, returning the fluorescent compound to its ground state. The emitted energy is lower than its excitation energy (light or laser) and thus of a longer wavelength. This shift or difference in energy or wavelength allows the emission energy to be detected and isolated from the excitation energy.
Conventional fluorescence detection typically utilizes wavelength filtering to isolate the emission photons from the excitation photons, and a detector that registers emission photons and produces a recordable output, usually as an electrical signal or a photographic image. However, several problems exist with conventional fluorescent detection techniques. For instance, most biological fluids possess autofluorescence that can decrease detection accuracy. The assay device may also possess some autofluorescence. These interferences are enhanced by the small Stokes shifts of many conventional fluorescent labels, e.g., between 20 to 50 nanometers.
In response to some of the problems with conventional fluorescence detection techniques, a technique known as “time-resolved” fluorescence was developed. Time-resolved fluorescence involves exciting the fluorescent label with a short pulse of light, then waiting a certain time (e.g., between approximately 100 to 200 microseconds) after excitation before measuring the remaining long-lived fluorescent signal. In this manner, any short-lived fluorescent background signals and scattered excitation radiation are eliminated. Although “time-resolved” techniques have been successfully employed in some types of assay devices, such as cuvette-based instruments, problems nevertheless remain in incorporating time-resolved techniques in other types of assay devices, such as membrane-based devices.
In particular, conventional time-resolved systems, such as those based on monochromators, involve very complex and expensive instruments. For example, a typical research-grade laboratory fluorimeter is a dual monochromator system, with one monochromator used to select the excitation wavelength and another monochromator used to select the detection wavelength. This level of complexity drastically increases the costs of the system and also requires a bulky, non-portable, and heavy instrument. In addition, conventional time-resolved systems are also problematic when used in conjunction with membrane-based assay devices. Specifically, in a membrane-based device, the concentration of the analyte is reduced because it is diluted by a liquid that can flow through the porous membrane. Unfortunately, background interference becomes increasingly problematic at such low analyte concentrations because the fluorescent intensity to be detected is relatively low. Because the structure of the membrane also tends to reflect the emitted light, the ability of the detector to accurately measure the fluorescent intensity of the labeled analyte is substantially reduced. In fact, the intensity of the emitted fluorescence signal is typically three to four orders of magnitude smaller than the excitation light reflected by the porous membrane.
As such, a need currently exists for a simple, inexpensive, and effective system for measuring the fluorescence in a membrane-based assay device.